Antibodies

ABSTRACT

Antibodies having modified constant regions so as to permit conjugation of the antibody to a payload such as a therapeutic agent are described. Preferred antibodies include a mutation at light chain position 180 (positional numbering), most preferably the mutation is to a residue selected from C, K, Q, or a non-natural amino acid. Additional mutations may also be combined with a mutation at position 180; including one or more of light chain (LC) S208, LC S171, LC S182, LC A184, LC V191, LC S202, LC S203, LC T206, heavy chain (HC) S160, HC T190, HC S443, HC S447, HC S139, HC S168, HC V170, HC V176, HC T200, HC S445 according to a positional numbering convention.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a U.S. National Phase of International PatentApplication No. PCT/EP2016/053162 filed 15 Feb. 2016, which claims thebenefit of priority to United Kingdom Patent Application, 1504858.0filed Mar. 23, 2015, and United Kingdom Patent Application 1502591.9filed Feb. 16, 2015, the contents of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies which include a modifiedconstant region so as to permit conjugation of the antibody to a payloadsuch as a radionuclide, a polymer, a cytotoxic agent or a drug moiety.Preferably the conjugated antibodies yield a desired payload to antibodyratio. Aspects of the invention relate to payload-antibody conjugates.

BACKGROUND TO THE INVENTION

Antibody-drug conjugates (ADCs) are a class of targeted therapies whichcombine the specificity of antibodies with the cytotoxicity of cytotoxictherapeutics. ADCs are primarily considered candidates for the treatmentof various cancers. ADCs comprise an antibody linked to a therapeuticdrug.

One problem with development of ADCs is the conjugation technology used;if drugs are conjugated non-selectively to cysteine or lysine residuesin the antibody, then this can result in a heterogeneous mixture ofADCs. This approach leads to suboptimal safety and efficacy propertiesand makes optimization of the biological, physical and pharmacologicalproperties of an ADC challenging. In particular, heterogeneity can be aproblem with respect to the distribution of cytotoxins (that is, site ofattachment), and the loading of cytotoxins (that is, number of drugmolecules per antibody).

Heterogeneity presents safety concerns since high drug/antibody ratio(DAR) species can have poor binding to their target and increase risksof off-target toxicity. Low drug loading species are less active (DAR 1)or inactive (DAR 0). As the number of drugs per mAb decreases, thepharmacokinetic properties of the ADC improves (Hamblett, Clin. CancerRes. 2004, 10, 7063-7070). Furthermore, heterogeneity of ADCs leads tochallenges associated with consistent manufacturing and analyticaltesting.

Site-selective conjugation (SSC) would presumably improve ADCs' safetyand efficacy, and thus, results in higher ADC quality. Junutula et alNature Biotech 2008 report that same activity is achieved with half ADCdose in a SSC-ADC compared to control. Thus, ADC homogeneity willimprove Therapeutic Index (TI is the ratio between maximum tolerateddose and effective dose. (TI=TD50/ED50)). Furthermore, higher DARhomogeneity would result in:

-   -   Simpler product characterization for regulatory filings, better        defined product specifications and    -   Reduced off-target or bystander toxicity    -   Potentially more controlled pharmacology (fewer species to        reckon on)    -   Potentially reduce dosage requirements (per gram) to achieve        similar pharmacological effect    -   Reduced potential incidence of side-effects (e.g.        immunogenicity, toxicity, etc.)    -   Reduced costs due to higher conjugation yields and reduced        dosage (required amount of ADC to be administered)

However, simply making antibodies with modified residues may result inunforeseen effects, such as changes to antibody aggregation propensity,solubility, or efficacy. The present applicants therefore developed arational screening process to determine which residues may be modified.In selected embodiments, the modification achieves an antibody suitablefor conjugation to a payload (such as a drug) to give a DAR of around 2.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided anantibody, or a fragment or derivative thereof, having a variable regionwhich binds a target molecule, and a constant region, wherein theconstant region comprises one or more mutations introducing a sitespecific conjugation site selected so as to permit conjugation of theantibody, fragment, or derivative to a payload, wherein the one or moremutations include a mutation at light chain position 180 (positionalnumbering).

The antibody may comprise two introduced site specific conjugationsites.

In preferred embodiments, the wild type residue at position 180 is T.The mutated residue at position 180 is preferably selected fromcysteine, lysine, glutamine, or a non-natural amino acid, and is mostpreferably cysteine.

In certain embodiments, the antibody may further comprise one or moremutations selected from light chain (LC) S208, LC S171, LC S182, LCA184, LC V191, LC S202, LC S203, LC T206, heavy chain (HC) S160, HCT190, HC S443, HC S447, HC S139, HC S168, HC V170, HC V176, HC T200, HCS445 according to a positional numbering convention. Preferred mutationsare at one or more positions selected from residues 206 (light chain),160, 190, 443, and 447 (heavy chain) (positional numbering). The mutatedresidues are preferably independently selected from cysteine, lysine,glutamine, or a non-natural amino acid, and is most preferably cysteine.For example, the modification of antibodies with non-natural amino acidsis described in WO2013/185115.

According to a further aspect of the invention, there is provided anantibody, or a fragment or derivative thereof, having a variable regionwhich binds a target molecule, and a constant region, wherein theconstant region comprises one or more mutations introducing a sitespecific conjugation site selected so as to permit conjugation of theantibody, fragment, or derivative to a payload, wherein the antibodycomprises the amino acid sequence of SEQ ID No 33; where X is selectedfrom C, K, Q, or a non-natural amino acid. The antibody may furthercomprise one or more amino acid sequences selected from the groupconsisting of SEQ ID No 22-32 and SEQ ID No 34-40; where each X isindependently selected from C, K, Q, or a non-natural amino acid, and ismost preferably cysteine.

According to a second aspect of the invention, there is provided anantibody, or a fragment or derivative thereof, having a variable regionwhich binds a target molecule, and a constant region, wherein theconstant region comprises one or more mutations introducing a sitespecific conjugation site selected so as to permit conjugation of theantibody, fragment, or derivative to a payload, wherein the one or moremutations include a mutation at light chain position 208 (positionalnumbering).

The antibody may comprise two introduced site specific conjugationsites.

In preferred embodiments, the wild type residue at position 208 is S.The mutated residue at position 208 is preferably selected fromcysteine, lysine, glutamine, or a non-natural amino acid, and is mostpreferably cysteine.

In certain embodiments, the antibody may further comprise one or moremutations selected from light chain (LC) T180, LC S171, LC S182, LCA184, LC V191, LC S202, LC S203, LC T206, heavy chain (HC) S160, HCT190, HC S443, HC S447, HC S139, HC S168, HC V170, HC V176, HC T200, HCS445 according to a positional numbering convention. Preferred mutationsare at one or more positions selected from residues 180, 206 (lightchain), 160, 190, 443, and 447 (heavy chain) (positional numbering). Themutated residues are preferably independently selected from cysteine,lysine, glutamine, or a non-natural amino acid, and is most preferablycysteine. For example, the modification of antibodies with non-naturalamino acids is described in WO2013/185115.

According to a further aspect of the invention, there is provided anantibody, or a fragment or derivative thereof, having a variable regionwhich binds a target molecule, and a constant region, wherein theconstant region comprises one or more mutations introducing a sitespecific conjugation site selected so as to permit conjugation of theantibody, fragment, or derivative to a payload, wherein the antibodycomprises the amino acid sequence of SEQ ID No 40; where X is selectedfrom C, K, Q, or a non-natural amino acid. The antibody may furthercomprise one or more amino acid sequences selected from the groupconsisting of SEQ ID No 22-39; where each X is independently selectedfrom C, K, Q, or a non-natural amino acid, and is most preferablycysteine.

The invention further provides an isolated or engineered antibody, or afragment or derivative thereof, having a variable region which binds atarget molecule, and a constant region, wherein the constant regioncomprises one or more mutations introducing a site specific conjugationsite by:

-   -   a) substituting a residue selected from the group consisting of        light chain (LC) T180, LC S208, LC S171, LC S182, LC A184, LC        V191, LC S202, LC S203, LC T206, heavy chain (HC) S160, HC T190,        HC S443, HC S447, HC S139, HC S168, HC V170, HC V176, HC T200,        HC S445 according to a positional numbering convention with a        cysteine residue, a lysine residue, a glutamine residue or a        non-natural amino acid; or    -   b) introducing a cysteine substitution at a residue selected        from the group consisting of LC T180C, LC S208C, LC S171C, LC        S182C, LC A184C, LC V191C, LC S202C, LC S203C, LC T206C, HC        S160C, HC T190C, HC S443C, HC S447C, HC S139C, HC S168C, HC        V170C, HC V176C, HC T200C, HC S445C according to a positional        numbering convention; or    -   c) comprising at least one amino acid sequence selected from the        group consisting of: SEQ ID No 22 to 40, wherein X is a cysteine        residue, a lysine residue, a glutamine residue or a non-natural        amino acid; or    -   d) comprising at least one amino acid sequence selected from the        group consisting of: SEQ ID No 22 to 40, wherein X is a cysteine        residue.

The invention still further provides an antibody, or a fragment orderivative thereof, having a variable region which binds a targetmolecule, and a constant region, wherein the constant region comprisesone or more mutations introducing a site specific conjugation siteselected so as to permit conjugation of the antibody, fragment, orderivative to a payload, wherein the antibody includes a light chainconstant region comprising the amino acid sequence of residues 109-214of SEQ ID No 14.

The invention also provides an antibody, or a fragment or derivativethereof, having a variable region which binds a target molecule, and aconstant region, wherein the constant region comprises one or moremutations introducing a site specific conjugation site selected so as topermit conjugation of the antibody, fragment, or derivative to apayload, wherein the antibody includes a light chain constant regioncomprising the amino acid sequence of residues 109-214 of SEQ ID No 21.

The following features may apply to all aspects of the invention. Theantibody is preferably selected from the group comprising IgG1, IgG2,IgG3, and IgG4. The light chain may either be kappa or lambda lightchain, the light chain constant region either C kappa (Ck) or C lambda(Cλ). The constant region may comprise at least a portion of an IgG1constant region, preferably one or more, preferably all, of the Ck, CH1and CH3 domains of the IgG1 constant region. The antibody may beselected from the group consisting of Fabs, bi specific antibodyfragments (tandem scFv-Fc, scFv-Fc knobs-into-holes, scFv-Fc-scFv,F(ab′)2, Fab-scFv, (Fab′scFv)2, scDiabody-Fc, or scDiabody-CH3),IgG-based bispecific antibodies (Hybrid hybridoma, Knobs-into-holes withcommon light chain, Two-in-one IgG, Dual V domain IgG, IgG-scFv,scFv-IgG, IgG-V, V-IgG), minibody, tribi-minibody, nanobodies, anddi-diabody. The antibody may be human, humanised, or chimeric.

Preferred antibodies are selected from Abciximab; Rituximab;Basiliximab; Daclizumab; Palivizumab; Infliximab; Trastuzumab;Alemtuzumab; Adalimumab; Efalizumab; Cetuximab; Ibritumomab; Omalizumab;Bevacizumab; Ranibizumab; Golimumab; Canakinumab; Ustekinumab;Tocilizumab; Ofatumumab; Belimumab; Ipilimumab; Brentuximab; Pertuzumab;Raxibacumab; Vedolizumab; Ramucirumab; Obinutuzumab; Siltuximab;Secukinumab; Dinutuximab.

The antibody may lack one or more Fc effector functions; may lack ADCCactivity; or may have increased ADCC activity.

A further aspect of the invention provides an antibody, or a fragment orderivative thereof, having a variable region which binds a targetmolecule, and a constant region, wherein the constant region comprisesone or more mutations introducing a site specific conjugation siteselected so as to permit conjugation of the antibody, fragment, orderivative to a payload, wherein the antibody includes a light chaincomprising the amino acid sequence of SEQ ID No 14. The antibody mayinclude a heavy chain comprising an amino acid sequence selected fromSEQ ID No 2 to SEQ ID No 12.

In another aspect of the invention provides an antibody, or a fragmentor derivative thereof, having a variable region which binds a targetmolecule, and a constant region, wherein the constant region comprisesone or more mutations introducing a site specific conjugation siteselected so as to permit conjugation of the antibody, fragment, orderivative to a payload, wherein the antibody includes a light chaincomprising the amino acid sequence of SEQ ID No 21. The antibody mayinclude a heavy chain comprising an amino acid sequence selected fromSEQ ID No 2 to SEQ ID No 12.

The invention further provides an immunoconjugate comprising an antibodyaccording to any of the preceding aspects of the invention, a payload,and a linker joining the payload to the antibody. The linker may beselected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP),valine-citrulline (val-cit), alanine-phenylalanine (ala-phe),p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1carboxylate (SMCC), N-Succinimidyl, (4-iodo-acetyl) aminobenzoate(SIAB), and 6-maleimidocaproyl-valine-citrulline-p-aminobenyloxycarbonyl(MC-vc-PAB); or is a branched linker which comprises a peptide chain andis derived from o-hydroxy p-amino benzylic alcohol, wherein the peptidechain is connected to the phenyl ring via the p-amino group, the payloadis connected to the phenyl ring via the benzylic alcohol moiety, and theantibody is connected to the phenyl ring via the o-hydroxy group.

The payload may be selected from the group consisting of a radionuclide,a chemotherapeutic agent, a cytotoxic agent, a microbial toxin, a planttoxin, a polymer, a carbohydrate, a cytokine, a fluorescent label, aluminescent label, an enzyme-substrate label, an enzyme, a peptide, apeptidomimetic, a nucleotide, an siRNA, a microRNA, an RNA mimetic, andan aptamer. A preferred polymer is a PEG molecule. Other preferredpayloads include 90Y, 131I, 67Cu, 177Lu, 213Bi, 211At, dolastatin,vedotin, monomethyl auristatin F, maytansinoids including DM1 and DM4,duocarmycin analogs, calicheamicin, pyrrolobenzodiazepines,centanamycin, irinotecan, and doxorubicin, Pseudomonas exotoxin A,Diphtheria toxin, ricin, polyethylene glycol, hydroxyethyl starch, and amannosyl residue. The payload may be a microtubule disrupting agent, ora DNA modifying agent.

The invention further provides a pharmaceutical composition comprisingan antibody or an immunoconjugate according to any of the above aspects,and a pharmaceutically acceptable diluent, carrier or excipient.

Also provided is a method for generating an immunoconjugate, the methodcomprising conjugating an antibody according to any of the above aspectsto a payload.

A process for preparing an antibody may comprise:

(i) mutagenizing a nucleic acid sequence of a parent antibody byreplacing one or more amino acid residues with a mutant residue toencode the antibody;

(ii) expressing the mutant antibody; and

(iii) isolating the mutant antibody.

The antibody may be expressed in a prokaryotic (such as E.coli orBacillus) or an eukaryotic cell (such as yeast (Pichia, Saccharomyces,Hansenula, Yarrowia) or mammalian cells (CHO cells, NSO cells, SP2/0cells, 293 cells) or insect cells (SF9 cells).

The process may further comprise:

(i) reacting the mutant antibody with a thiol-reactive affinity reagentto generate an affinity labelled, antibody; and

(ii) measuring the binding of the affinity labelled antibody to acapture media.

Other aspects of the invention provide an antibody, or a fragment orderivative thereof, having a variable region which binds a targetmolecule, and a constant region, wherein the constant region comprisesone or more mutations introducing a site specific conjugation siteselected so as to permit conjugation of the antibody, fragment, orderivative to a payload, such that from 85% to 110% of site specificconjugation sites per antibody are conjugated to a payload. In apreferred embodiment at least 90%, at least 95%, or at least 100% ofsite specific conjugation sites per antibody are conjugated to apayload. In a preferred embodiment, the antibody comprises two sitespecific conjugation sites (ie, one on either the heavy or light chain,and two such chains per antibody). For such an antibody, 85-110%conjugation yields a drug-antibody ratio (DAR) of from 1.7-2.2. Incertain embodiments, the DAR is preferably from 1.8-2.1, more preferablyfrom 1.9-2.1, and most preferably around 2.0.

In another preferred embodiment, the antibody comprises four sitespecific conjugation sites (ie, one on the heavy and one on the lightchain, or two on the heavy and none on the light chain, or none on theheavy and two on the light chain, and two such chains per antibody). Forsuch an antibody, 85-110% conjugation yields a drug-antibody ratio (DAR)of from 2×(1.7-2.2) i.e. 3.4-4.4. In certain embodiments, the DAR ispreferably from 2×1.8-2.1, i.e. 3.6-4.2, more preferably from 2×1.9-2.1i.e. 3.8-4.2, and most preferably around 2×2.0 i.e. 4.

The antibody, fragment, or derivative may be conjugated directly orindirectly to the payload; indirect conjugation may take place via alinker. The antibody may further comprise a linker. Suitable linkersinclude maleimide linkers, which permit conjugation to a payload viasuccinimide conjugation. The linker may be cleavable, or non-cleavable.Other suitable linkers are disclosed in international patent applicationWO2012/113847, which describes a branched linker which comprises apeptide chain and is derived from o-hydroxy p-amino benzylic alcohol,wherein the peptide chain is connected to the phenyl ring via thep-amino group, the drug is connected to the phenyl ring via the benzylicalcohol moiety, and the antibody is connected to the phenyl ring via theo-hydroxy group.

The constant region preferably comprises at least a portion of an IgGIgA, IgD, IgE, or IgM constant region, more preferably a human IgGconstant region. In preferred embodiments, the constant region is a fulllength IgG constant region, although in other embodiments truncatedconstant regions or light or heavy chain only constant regions may beused. For example, the constant region may comprise one or more,preferably all, of the Ck, CH1 and CH3 domains of the IgG constantregion. A preferred IgG is IgG1, other IgG include IgG2, IgG3, and IgG4.

By “antibody” is meant any antigen-binding immunoglobulin molecule. Theantibody is preferably a complete mammalian antibody (comprising twoheavy chains and two light chains, each of which includes a constantregion and a variable region), but other forms of antibody andderivative may be used. For example, Fabs, bi specific antibodyfragments (tandem scFv-Fc, scFv-Fc knobs-into-holes, scFv-Fc-scFv,F(ab′)₂, Fab-scFv, (Fab′scFv)₂, scDiabody-Fc, or scDiabody-C_(H)3),IgG-based bispecific antibodies (Hybrid hybridoma, Knobs-into-holes withcommon light chain, Two-in-one IgG, Dual V domain IgG, IgG-scFv,scFv-IgG, IgG-V, V-IgG), minibody, tribi-minibody, nanobodies,di-diabody.

The antibody may be human, or humanised, or chimeric.

The one or more mutations preferably comprise mutation from anon-cysteine amino acid to a cysteine amino acid. Preferably thenon-cysteine amino acid is selected from serine, valine, threonine, oralanine; more preferably serine or threonine. Alternatively, themutation may be to lysine, glutamine, or a non-natural amino acid.

In preferred embodiments, the one or more mutations is selected fromS160C, T190C, S443C, S447C (on the heavy chain), T180C,or T260C (on thelight chain). The numbering recited herein is positional numbering basedon the full length trastuzumab sequence. Other numbering conventions aresummarised in the below table:

IMGT ADC Strands, unique CH1/CL(kappa) Substitution turns and numberingIGHG1 (positional loops for C- for C- amino acid IMGT exon Eu Kabatnumbering) DOMAINs DOMAINs translation numbering numbering numbering H:S160C C-strand 40 S 40 157 156 H: T190C E-strand 90 T 70 187 192 H:S443C G-strand 120 S 100 440 471 H: S447C G-strand 124 S 104 444 475 L:T180C E-strand 90 T 73 180 180 L: T206C G-strand 118 T 99 206 206

Preferred antibodies include the light chain T180 mutation. This may bethe sole mutation, or may be combined with any of the other mutationsdisclosed herein.

Preferred antibodies comprise combinations of 2 mutations which areselected from the group of

LC T180 in combination with LC S208,

LC T180 in combination with LC S171,

LC T180 in combination with LC S182,

LC T180 in combination with LC A184,

LC T180 in combination with LC V191,

LC T180 in combination with LC S202,

LC T180 in combination with LC S203,

LC T180 in combination with LC T206,

LC T180 in combination with HC S160,

LC T180 in combination with HC T190

LC T180 in combination with HC S443,

LC T180 in combination with HC S447,

LC T180 in combination with HC S139,

LC T180 in combination with HC S168,

LC T180 in combination with HC V170,

LC T180 in combination with HC V176,

LC T180 in combination with HC T200,

LC T180 in combination with HC S445.

The mutations are preferably mutations to cysteine, lysine, glutamine,or a non-natural amino acid.

Further preferred antibodies comprise combinations of 2 mutationsselected from the group of

LC T180C in combination with LC S208C,

LC T180C in combination with LC S171C,

LC T180C in combination with LC S182C,

LC T180C in combination with LC A184C,

LC T180C in combination with LC V191 C,

LC T180C in combination with LC S202C,

LC T180C in combination with LC S203C,

LC T180C in combination with LC T206C,

LC T180C in combination with HC S160C,

LC T180C in combination with HC T190C,

LC T180C in combination with HC S430C,

LC T180C in combination with HC S447C,

LC T180C in combination with HC S139C,

LC T180C in combination with HC S168C,

LC T180C in combination with HC V170C,

LC T180C in combination with HC V176C,

LC T180C in combination with HC T200C,

LC T180C in combination with HC S445C.

Most preferred antibodies comprise LC T180C in combination with HCS160C.

Other preferred antibodies comprise combinations of the 2 amino acidsequences selected from the group of:

SEQ ID No. 33 and SEQ ID No. 22,

SEQ ID No. 33 and SEQ ID No. 23,

SEQ ID No. 33 and SEQ ID No. 24,

SEQ ID No. 33 and SEQ ID No. 25,

SEQ ID No. 33 and SEQ ID No. 26,

SEQ ID No. 33 and SEQ ID No. 27,

SEQ ID No. 33 and SEQ ID No. 28,

SEQ ID No. 33 and SEQ ID No. 29,

SEQ ID No. 33 and SEQ ID No. 30,

SEQ ID No. 33 and SEQ ID No. 31,

SEQ ID No. 33 and SEQ ID No. 32,

SEQ ID No. 33 and SEQ ID No. 34,

SEQ ID No. 33 and SEQ ID No. 35,

SEQ ID No. 33 and SEQ ID No. 36,

SEQ ID No. 33 and SEQ ID No. 37,

SEQ ID No. 33 and SEQ ID No. 38,

SEQ ID No. 33 and SEQ ID No. 39,

SEQ ID No. 33 and SEQ ID No. 40.

In preferred antibodies, X is cysteine. Alternatively, X may beindependently selected from lysine, glutamine, cysteine, or anon-natural amino acid, and is most preferably cysteine.

Most preferred antibodies comprise SEQ ID No. 33 and SEQ ID No. 23.

Additional mutations which may be combined with any of the aspectsdisclosed herein include:

Engineered SEQ Cysteine Cys (Positional ID (Kabat) No.) Sequence No:Heavy chain S134C S139 FPLAPSSKST X GGTAALGCLV 22 S156C S160 KDYFPEPVTVX WNSGALTSGV 23 S168C S168 TVSWNSGALT X GVHTFPAVLQ 24 V171C V170SWNSGALTSG X HTFPAVLQSS 25 V177C V176 LTSGVHTFPA X LQSSGLYSLS 26 T192CT190 SGLYSLSSVV X VPSSSLGTQT 27 T205C T200 TVPSSSLGTQ X YICNVNHKPS 28S471C S443 EALHNHYTQK X LSLSPGK 29 S473C S445 EALHNHYTQKSL X LSPGK 30S475C S447 EALHNHYTQKSLSL X PGK 31 Light chain S171C S171 ESVTEQDSKD XTYSLSSTLTL 32 T180C T180 DSTYSLSSTL X LSKADYEKHK 33 S182C S182TYSLSSTLTL X KADYEKHKVY 34 A184C A184 SLSSTLTLSK X DYEKHKVYAC 35 V191CV191 LSKADYEKHK X YACEVTHQGL 36 S202C S202 YACEVTHQGL X SPVTKSFNRG 37S203C S203 ACEVTHQGLS X PVTKSFNRGE 38 T206C T206 VTHQGLSSPV X KSFNRGEC39 S208C S208 VTHQGLSSPVTK X FNRGEC 40

In the above table, the mutated residues are indicated as X. X ispreferably cysteine, but other mutant residues may be used, inparticular lysine, glutamine, or a non-natural amino acid. Note thatV191 may be L191 in certain parent antibodies, in particular Kappaallotypes Km1 and Km2; where reference is made herein to V191 mutations,then unless the context requires otherwise this is understood to referalso to L191 mutations.

Other preferred antibodies include the light chain T206 mutation incombination with one or more other mutations. In preferred embodiments,the light chain T206 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC T206 in combination with LC S208,

LC T206 in combination with LC S171,

LC T206 in combination with LC S182,

LC T206 in combination with LC A184,

LC T206 in combination with LC V191,

LC T206 in combination with LC S202,

LC T206 in combination with LC S203,

LC T206 in combination with LC T180,

LC T206 in combination with HC S160,

LC T206 in combination with HC T190,

LC T206 in combination with HC S443,

LC T206 in combination with HC S447,

LC T206 in combination with HC S139,

LC T206 in combination with HC S168,

LC T206 in combination with HC V170,

LC T206 in combination with HC V176,

LC T206 in combination with HC T200,

LC T206 in combination with HC S445.

Of which are preferred

LC T206 in combination with LC T180,

LC T206 in combination with HC S160,

LC T206 in combination with HC T190,

LC T206 in combination with HC S443,

LC T206 in combination with HC S447,

Of which are most preferred

LC T206C in combination with LC T180C,

LC T260C in combination with HC S160C,

LC T206C in combination with HC T190C

LC T206C in combination with HC S443C,

LC T260C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 39 together with an amino acid sequence selectedfrom SEQ ID No 22 to 38 or 40. In preferred antibodies, X is cysteine.Alternatively, X may be independently selected from lysine, glutamine,cysteine, or a non-natural amino acid, and is most preferably cysteine.

Other preferred antibodies include the heavy chain S160 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain S160 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC S160 in combination with LC S208,

HC S160 in combination with LC S171,

HC S160 in combination with LC S182,

HC S160 in combination with LC A184,

HC S160 in combination with LC V191,

HC S160 in combination with LC S202,

HC S160 in combination with LC S203,

HC S160 in combination with LC T180,

HC S160 in combination with LC T206,

HC S160 in combination with HC T190,

HC S160 in combination with HC S443,

HC S160 in combination with HC S447,

HC S160 in combination with HC S139,

HC S160 in combination with HC S168,

HC S160 in combination with HC V170,

HC S160 in combination with HC V176,

HC S160 in combination with HC T200,

HC S160 in combination with HC S445.

Of which are preferred

HC S160 in combination with LC T180,

HC S160 in combination with LC T206,

HC S160 in combination with HC T190,

HC S160 in combination with HC S443,

HC S160 in combination with HC S447,

Of which are most preferred

HC S160C in combination with LC T180C,

HC S160C in combination with LC T206C,

HC S160C in combination with HC T190C,

HC S160C in combination with HC S443C,

HC S160C in combination with HC S447C,

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 23 together with an amino acid sequence selectedfrom SEQ ID No 22 or 24 to 40. In preferred antibodies, X is cysteine.Alternatively, X may be independently selected from lysine, glutamine,cysteine, or a non-natural amino acid, and is most preferably cysteine.

Other preferred antibodies include the heavy chain T190 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain T190 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC T190 in combination with LC S208,

HC T190 in combination with LC S171,

HC T190 in combination with LC S182,

HC T190 in combination with LC A184,

HC T190 in combination with LC V191,

HC T190 in combination with LC S202,

HC T190 in combination with LC S203,

HC T190 in combination with LC T180,

HC T190 in combination with LC T206,

HC T190 in combination with HC S160,

HC T190 in combination with HC S443,

HC T190 in combination with HC S447,

HC T190 in combination with HC S139,

HC T190 in combination with HC S168,

HC T190 in combination with HC V170,

HC T190 in combination with HC V176,

HC T190 in combination with HC T200,

HC T190 in combination with HC S445.

Of which are preferred

HC T190 in combination with LC T180,

HC T190 in combination with LC T206,

HC T190 in combination with HC S160,

HC T190 in combination with HC S443,

HC T190 in combination with HC S447,

Of which are most preferred

HC T1900 in combination with LC T180C,

HC T1900 in combination with LC T206C,

HC T1900 in combination with HC S160C,

HC T1900 in combination with HC S443C,

HC T1900 in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 27 together with an amino acid sequence selectedfrom SEQ ID No 22 to 26 or 28 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain S443 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain S443 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC S443 in combination with LC S208,

HC S443 in combination with LC S171,

HC S443 in combination with LC S182,

HC S443 in combination with LC A184,

HC S443 in combination with LC V191,

HC S443 in combination with LC S202,

HC S443 in combination with LC S203,

HC S443 in combination with LC T180,

HC S443 in combination with LC T206,

HC S443 in combination with HC S160,

HC S443 in combination with HC T190,

HC S443 in combination with HC S447,

HC S443 in combination with HC S139,

HC S443 in combination with HC S168,

HC S443 in combination with HC V170,

HC S443 in combination with HC V176,

HC S443 in combination with HC T200,

HC S443 in combination with HC S445.

Of which are preferred

HC S443 in combination with LC T180,

HC S443 in combination with LC T206,

HC S443 in combination with HC S160,

HC S443 in combination with HC T190,

HC S443 in combination with HC S447,

Of which are most preferred

HC S443C in combination with LC T180C,

HC S443C in combination with LC T206C,

HC S443C in combination with HC S160C,

HC S443C in combination with HC T190C,

HC S443C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 29 together with an amino acid sequence selectedfrom SEQ ID No 22 to 28 or 30 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain S447 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain S447 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC S447 in combination with LC S208,

HC S447 in combination with LC S171,

HC S447 in combination with LC S182,

HC S447 in combination with LC A184,

HC S447 in combination with LC V191,

HC S447 in combination with LC S202,

HC S447 in combination with LC S203,

HC S447 in combination with LC T180,

HC S447 in combination with LC T206,

HC S447 in combination with HC S160,

HC S447 in combination with HC T190,

HC S447 in combination with HC S443,

HC S447 in combination with HC S139,

HC S447 in combination with HC S168,

HC S447 in combination with HC V170,

HC S447 in combination with HC V176,

HC S447 in combination with HC T200,

HC S447 in combination with HC S445.

Of which are preferred

HC S447 in combination with LC T180,

HC S447 in combination with LC T206,

HC S447 in combination with HC S160,

HC S447 in combination with HC T190,

HC S447 in combination with HC S443,

Of which are most preferred

HC S447C in combination with LC T180C,

HC S447C in combination with LC T206C,

HC S447C in combination with HC S160C,

HC S447C in combination with HC T190C,

HC S447C in combination with HC S443C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 31 together with an amino acid sequence selectedfrom SEQ ID No 22 to 30 or 32 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the light chain S208 mutation incombination with one or more other mutations. In preferred embodiments,the light chain S208 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC S208 in combination with LC S171,

LC S208 in combination with LC S182,

LC S208 in combination with LC A184,

LC S208 in combination with LC V191,

LC S208 in combination with LC S202,

LC S208 in combination with LC S203,

LC S208 in combination with LC T180,

LC S208 in combination with LC T206,

LC S208 in combination with HC S160,

LC S208 in combination with HC T190,

LC S208 in combination with HC S443,

LC S208 in combination with HC S447,

LC S208 in combination with HC S139,

LC S208 in combination with HC S168,

LC S208 in combination with HC V170,

LC S208 in combination with HC V176,

LC S208 in combination with HC T200,

LC S208 in combination with HC S445.

Of which are preferred

LC S208 in combination with LC T180,

LC S208 in combination with LC T206,

LC S208 in combination with HC S160,

LC S208 in combination with HC T190,

LC S208 in combination with HC S443,

LC S208 in combination with HC S447,

Of which are most preferred

LC S208C in combination with LC T180C,

LC S208C in combination with LC T206C,

LC S208C in combination with HC S160C,

LC S208C in combination with HC T190C,

LC S208C in combination with HC S443C,

LC S208C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 40 together with an amino acid sequence selectedfrom SEQ ID No 22 to 39. In preferred antibodies, X is cysteine.Alternatively, X may be independently selected from lysine, glutamine,cysteine, or a non-natural amino acid, and is most preferably cysteine.

Accordingly, preferred antibodies comprise combinations of the 2 aminoacid sequences selected from the group of:

SEQ ID No. 40 and SEQ ID No. 22,

SEQ ID No. 40 and SEQ ID No. 23,

SEQ ID No. 40 and SEQ ID No. 24,

SEQ ID No. 40 and SEQ ID No. 25,

SEQ ID No. 40 and SEQ ID No. 26,

SEQ ID No. 40 and SEQ ID No. 27,

SEQ ID No. 40 and SEQ ID No. 28,

SEQ ID No. 40 and SEQ ID No. 29,

SEQ ID No. 40 and SEQ ID No. 30,

SEQ ID No. 40 and SEQ ID No. 31,

SEQ ID No. 40 and SEQ ID No. 32,

SEQ ID No. 40 and SEQ ID No. 33,

SEQ ID No. 40 and SEQ ID No. 34,

SEQ ID No. 40 and SEQ ID No. 35,

SEQ ID No. 40 and SEQ ID No. 36,

SEQ ID No. 40 and SEQ ID No. 37,

SEQ ID No. 40 and SEQ ID No. 38,

SEQ ID No. 40 and SEQ ID No. 39.

In preferred antibodies, X is cysteine. Alternatively, X may beindependently selected from lysine, glutamine, cysteine, or anon-natural amino acid, and is most preferably cysteine.

Other preferred antibodies include the light chain S171 mutation incombination with one or more other mutations. In preferred embodiments,the light chain S171 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC S171 in combination with LC S208,

LC S171 in combination with LC S182,

LC S171 in combination with LC A184,

LC S171 in combination with LC V191,

LC S171 in combination with LC S202,

LC S171 in combination with LC S203,

LC S171 in combination with LC T180,

LC S171 in combination with LC T206,

LC S171 in combination with HC S160,

LC S171 in combination with HC T190,

LC S171 in combination with HC S443,

LC S171 in combination with HC S447,

LC S171 in combination with HC S139,

LC S171 in combination with HC S168,

LC S171 in combination with HC V170,

LC S171 in combination with HC V176,

LC S171 in combination with HC T200,

LC S171 in combination with HC S445.

Of which are preferred

LC S171 in combination with LC T180,

LC S171 in combination with LC T206,

LC S171 in combination with HC S160,

LC S171 in combination with HC T190,

LC S171 in combination with HC S443,

LC S171 in combination with HC S447,

Of which are most preferred

LC S171C in combination with LC T180C,

LC S171C in combination with LC T206C,

LC S171C in combination with HC S160C,

LC S171C in combination with HC T190C,

LC S171C in combination with HC S443C,

LC S171C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 32 together with an amino acid sequence selectedfrom SEQ ID No 22 to 31 or 33 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the light chain S182 mutation incombination with one or more other mutations. In preferred embodiments,the light chain S182 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC S182 in combination with LC S208,

LC S182 in combination with LC S171,

LC S182 in combination with LC A184,

LC S182 in combination with LC V191,

LC S182 in combination with LC S202,

LC S182 in combination with LC S203,

LC S182 in combination with LC T180,

LC S182 in combination with LC T206,

LC S182 in combination with HC S160,

LC S182 in combination with HC T190,

LC S182 in combination with HC S443,

LC S182 in combination with HC S447,

LC S182 in combination with HC S139,

LC S182 in combination with HC S168,

LC S182 in combination with HC V170,

LC S182 in combination with HC V176,

LC S182 in combination with HC T200,

LC S182 in combination with HC S445.

Of which are preferred

LC S182 in combination with LC T180,

LC S182 in combination with LC T206,

LC S182 in combination with HC S160,

LC S182 in combination with HC T190,

LC S182 in combination with HC S443,

LC S182 in combination with HC S447,

Of which are most preferred

LC S182C in combination with LC T180C,

LC S182C in combination with LC T206C,

LC S182C in combination with HC S160C,

LC S182C in combination with HC T190C,

LC S182C in combination with HC S430C,

LC S182C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 34 together with an amino acid sequence selectedfrom SEQ ID No 22 to 33 or 35 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the light chain A184 mutation incombination with one or more other mutations. In preferred embodiments,the light chain A184 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC A184 in combination with LC S208,

LC A184 in combination with LC S171,

LC A184 in combination with LC S182,

LC A184 in combination with LC V191,

LC A184 in combination with LC S202,

LC A184 in combination with LC S203,

LC A184 in combination with LC T180,

LC A184 in combination with LC T206,

LC A184 in combination with HC S160,

LC A184 in combination with HC T190,

LC A184 in combination with HC S443,

LC A184 in combination with HC S447,

LC A184 in combination with HC S139,

LC A184 in combination with HC S168,

LC A184 in combination with HC V170,

LC A184 in combination with HC V176,

LC A184 in combination with HC T200,

LC A184 in combination with HC S445.

Of which are preferred

LC A184 in combination with LC T180,

LC A184 in combination with LC T206,

LC A184 in combination with HC S160,

LC A184 in combination with HC T190,

LC A184 in combination with HC S443,

LC A184 in combination with HC S447,

Of which are most preferred

LC A184C in combination with LC T180C,

LC A184C in combination with LC T206C,

LC A184C in combination with HC S160C,

LC A184C in combination with HC T190C,

LC A184C in combination with HC S443C,

LC A184C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 35 together with an amino acid sequence selectedfrom SEQ ID No 22 to 34 or 36 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the light chain V191 mutation incombination with one or more other mutations. In preferred embodiments,the light chain V191 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC V191 in combination with LC S208,

LC V191 in combination with LC S171,

LC V191 in combination with LC S182,

LC V191 in combination with LC A184,

LC V191 in combination with LC S202,

LC V191 in combination with LC S203,

LC V191 in combination with LC T180,

LC V191 in combination with LC T206,

LC V191 in combination with HC S160,

LC V191 in combination with HC T190,

LC V191 in combination with HC S443,

LC V191 in combination with HC S447,

LC V191 in combination with HC S139,

LC V191 in combination with HC S168,

LC V191 in combination with HC V170,

LC V191 in combination with HC V176,

LC V191 in combination with HC T200,

LC V191 in combination with HC S445.

Of which are preferred

LC V191 in combination with LC T180,

LC V191 in combination with LC T206,

LC V191 in combination with HC S160,

LC V191 in combination with HC T190,

LC V191 in combination with HC S443,

LC V191 in combination with HC S447,

Of which are most preferred

LC V191C in combination with LC T180C,

LC V191C in combination with LC T206C,

LC V191C in combination with HC S160C,

LC V191C in combination with HC T190C,

LC V191C in combination with HC S430C,

LC V191C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 36 together with an amino acid sequence selectedfrom SEQ ID No 22 to 35 or 37 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the light chain S202 mutation incombination with one or more other mutations. In preferred embodiments,the light chain S202 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC S202 in combination with LC S208,

LC S202 in combination with LC S171,

LC S202 in combination with LC S182,

LC S202 in combination with LC A184,

LC S202 in combination with LC V191,

LC S202 in combination with LC S203,

LC S202 in combination with LC T180,

LC S202 in combination with LC T206,

LC S202 in combination with HC S160,

LC S202 in combination with HC T190,

LC S202 in combination with HC S443,

LC S202 in combination with HC S447,

LC S202 in combination with HC S139,

LC S202 in combination with HC S168,

LC S202 in combination with HC V170,

LC S202 in combination with HC V176,

LC S202 in combination with HC T200,

LC S202 in combination with HC S445.

Of which are preferred

LC S202 in combination with LC T180,

LC S202 in combination with LC T206,

LC S202 in combination with HC S160,

LC S202 in combination with HC T190,

LC S202 in combination with HC S443,

LC S202 in combination with HC S447,

Of which are most preferred

LC S202C in combination with LC T180C,

LC S202C in combination with LC T206C,

LC S202C in combination with HC S160C,

LC S202C in combination with HC T190C,

LC S202C in combination with HC S430C,

LC S202C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 37 together with an amino acid sequence selectedfrom SEQ ID No 22 to 36 or 38 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the light chain S203 mutation incombination with one or more other mutations. In preferred embodiments,the light chain S203 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

LC S203 in combination with LC S208,

LC S203 in combination with LC S171,

LC S203 in combination with LC S182,

LC S203 in combination with LC A184,

LC S203 in combination with LC V191,

LC S203 in combination with LC S202,

LC S203 in combination with LC T180,

LC S203 in combination with LC T206,

LC S203 in combination with HC S160,

LC S203 in combination with HC T190,

LC S203 in combination with HC S443,

LC S203 in combination with HC S447,

LC S203 in combination with HC S139,

LC S203 in combination with HC S168,

LC S203 in combination with HC V170,

LC S203 in combination with HC V176,

LC S203 in combination with HC T200,

LC S203 in combination with HC S445.

Of which are preferred

LC S203 in combination with LC T180,

LC S203 in combination with LC T206,

LC S203 in combination with HC S160,

LC S203 in combination with HC T190,

LC S203 in combination with HC S443,

LC S203 in combination with HC S447,

Of which are most preferred

LC S203C in combination with LC T180C,

LC S203C in combination with LC T206C,

LC S203C in combination with HC S160C,

LC S203C in combination with HC T190C,

LC S203C in combination with HC S443C,

LC S203C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 38 together with an amino acid sequence selectedfrom SEQ ID No 22 to 37 or 39 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain S139 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain S139 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC S139 in combination with LC S208,

HC S139 in combination with LC S171,

HC S139 in combination with LC S182,

HC S139 in combination with LC A184,

HC S139 in combination with LC V191,

HC S139 in combination with LC S202,

HC S139 in combination with LC S203,

HC S139 in combination with LC T180,

HC S139 in combination with LC T206,

HC S139 in combination with HC S160,

HC S139 in combination with HC T190,

HC S139 in combination with HC S443,

HC S139 in combination with HC S447,

HC S139 in combination with HC S168,

HC S139 in combination with HC V170,

HC S139 in combination with HC V176,

HC S139 in combination with HC T200,

HC S139 in combination with HC S445.

Of which are preferred

HC S139 in combination with LC T180,

HC S139 in combination with LC T206,

HC S139 in combination with HC S160,

HC S139 in combination with HC T190,

HC S139 in combination with HC S443,

HC S139 in combination with HC S447,

Of which are most preferred

HC S139C in combination with LC T180C,

HC S139C in combination with LC T206C,

HC S139C in combination with HC S160C,

HC S139C in combination with HC T190C,

HC S139C in combination with HC S443C,

HC S139C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 22 together with an amino acid sequence selectedfrom SEQ ID No 23 to 40. In preferred antibodies, X is cysteine.Alternatively, X may be independently selected from lysine, glutamine,cysteine, or a non-natural amino acid, and is most preferably cysteine.

Other preferred antibodies include the heavy chain S168 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain S168 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC S168 in combination with LC S208,

HC S168 in combination with LC S171,

HC S168 in combination with LC S182,

HC S168 in combination with LC A184,

HC S168 in combination with LC V191,

HC S168 in combination with LC S202,

HC S168 in combination with LC S203,

HC S168 in combination with LC T180,

HC S168 in combination with LC T206,

HC S168 in combination with HC S160,

HC S168 in combination with HC T190,

HC S168 in combination with HC S443,

HC S168 in combination with HC S447,

HC S168 in combination with HC S139,

HC S168 in combination with HC V170,

HC S168 in combination with HC V176,

HC S168 in combination with HC T200,

HC S168 in combination with HC S445.

Of which are preferred

HC S168 in combination with LC T180,

HC S168 in combination with LC T206,

HC S168 in combination with HC S160,

HC S168 in combination with HC T190,

HC S168 in combination with HC S443,

HC S168 in combination with HC S447,

Of which are most preferred

HC S168C in combination with LC T180C,

HC S168C in combination with LC T206C,

HC S168C in combination with HC S160C,

HC S168C in combination with HC T190C,

HC S168C in combination with HC S443C,

HC S168C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 24 together with an amino acid sequence selectedfrom SEQ ID No 22, 23, or 25 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain V170 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain V170 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC V170 in combination with LC S208,

HC V170 in combination with LC S171,

HC V170 in combination with LC S182,

HC V170 in combination with LC A184,

HC V170 in combination with LC V191,

HC V170 in combination with LC S202,

HC V170 in combination with LC S203,

HC V170 in combination with LC T180,

HC V170 in combination with LC T206,

HC V170 in combination with HC S160,

HC V170 in combination with HC T190,

HC V170 in combination with HC S443,

HC V170 in combination with HC S447,

HC V170 in combination with HC S139,

HC V170 in combination with HC S168,

HC V170 in combination with HC V176,

HC V170 in combination with HC T200,

HC V170 in combination with HC S445.

Of which are preferred

HC V170 in combination with LC T180,

HC V170 in combination with LC T206,

HC V170 in combination with HC S160,

HC V170 in combination with HC T190,

HC V170 in combination with HC S443,

HC V170 in combination with HC S447,

Of which are most preferred

HC V170C in combination with LC T180C,

HC V170C in combination with LC T206C,

HC V170C in combination with HC S160C,

HC V170C in combination with HC T190C,

HC V170C in combination with HC S443C,

HC V170C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 25 together with an amino acid sequence selectedfrom SEQ ID No 22 to 24 or 26 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain V176 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain V176 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC V176 in combination with LC S208,

HC V176 in combination with LC S171,

HC V176 in combination with LC S182,

HC V176 in combination with LC A184,

HC V176 in combination with LC V191,

HC V176 in combination with LC S202,

HC V176 in combination with LC S203,

HC V176 in combination with LC T180,

HC V176 in combination with LC T206,

HC V176 in combination with HC S160,

HC V176 in combination with HC T190,

HC V176 in combination with HC S443,

HC V176 in combination with HC S447,

HC V176 in combination with HC S139,

HC V176 in combination with HC S168,

HC V176 in combination with HC V170,

HC V176 in combination with HC T200,

HC V176 in combination with HC S445.

Of which are preferred

HC V176 in combination with LC T180,

HC V176 in combination with LC T206,

HC V176 in combination with HC S160,

HC V176 in combination with HC T190,

HC V176 in combination with HC S443,

HC V176 in combination with HC S447,

Of which are most preferred

HC V176C in combination with LC T180C,

HC V176C in combination with LC T206C,

HC V176C in combination with HC S160C,

HC V176C in combination with HC T190C,

HC V176C in combination with HC S443C,

HC V176C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 26 together with an amino acid sequence selectedfrom SEQ ID No 22 to 25 or 27 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain T200 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain T200 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC T200 in combination with LC S208,

HC T200 in combination with LC S171,

HC T200 in combination with LC S182,

HC T200 in combination with LC A184,

HC T200 in combination with LC V191,

HC T200 in combination with LC S202,

HC T200 in combination with LC S203,

HC T200 in combination with LC T180,

HC T200 in combination with LC T206,

HC T200 in combination with HC S160,

HC T200 in combination with HC T190,

HC T200 in combination with HC S443,

HC T200 in combination with HC S447,

HC T200 in combination with HC S139,

HC T200 in combination with HC S168,

HC T200 in combination with HC V170,

HC T200 in combination with HC V176,

HC T200 in combination with HC S445.

Of which are preferred

HC T200 in combination with LC T180,

HC T200 in combination with LC T206,

HC T200 in combination with HC S160,

HC T200 in combination with HC T190,

HC T200 in combination with HC S443,

HC T200 in combination with HC S447,

Of which are most preferred

HC T200C in combination with LC T180C,

HC T200C in combination with LC T206C,

HC T200C in combination with HC S160C,

HC T200C in combination with HC T190C,

HC T200C in combination with HC S443C,

HC T200C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 28 together with an amino acid sequence selectedfrom SEQ ID No 22 to 27 or 29 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

Other preferred antibodies include the heavy chain S445 mutation incombination with one or more other mutations. In preferred embodiments,the heavy chain S445 mutation is combined with another mutationdescribed herein. The following are preferred combinations of twomutations; antibodies according to the invention may comprise thesemutations together with one or more additional mutations.

HC S445 in combination with LC S208,

HC S445 in combination with LC S171,

HC S445 in combination with LC S182,

HC S445 in combination with LC A184,

HC S445 in combination with LC V191,

HC S445 in combination with LC S202,

HC S445 in combination with LC S203,

HC S445 in combination with LC T180,

HC S445 in combination with LC T206,

HC S445 in combination with HC S160,

HC S445 in combination with HC T190,

HC S445 in combination with HC S443,

HC S445 in combination with HC S447,

HC S445 in combination with HC S139,

HC S445 in combination with HC S168,

HC S445 in combination with HC V170,

HC S445 in combination with HC V176,

HC S445 in combination with HC T200.

Of which are preferred

HC S445 in combination with LC T180,

HC S445 in combination with LC T206,

HC S445 in combination with HC S160,

HC S445 in combination with HC T190,

HC S445 in combination with HC S443,

HC S445 in combination with HC S447,

Of which are most preferred

HC S445C in combination with LC T180C,

HC S445C in combination with LC T206C,

HC S445C in combination with HC S160C,

HC S445C in combination with HC T190C,

HC S445C in combination with HC S443C,

HC S445C in combination with HC S447C.

Thus, preferred antibodies may comprise combinations of the amino acidsequence of SEQ ID No 30 together with an amino acid sequence selectedfrom SEQ ID No 22 to 29 or 31 to 40. In preferred antibodies, X iscysteine. Alternatively, X may be independently selected from lysine,glutamine, cysteine, or a non-natural amino acid, and is most preferablycysteine.

The antibody may be selected from Abciximab; Rituximab; Basiliximab;Daclizumab; Palivizumab; Infliximab; Trastuzumab; Alemtuzumab;Adalimumab; Efalizumab; Cetuximab; Ibritumomab; Omalizumab; Bevacizumab;Ranibizumab; Golimumab; Canakinumab; Ustekinumab; Tocilizumab;Ofatumumab; Belimumab; Ipilimumab; Brentuximab; Pertuzumab; Raxibacumab;Vedolizumab; Ramucirumab; Obinutuzumab; Siltuximab; Secukinumab;Dinutuximab.

The invention further provides an antibody having a light chaincomprising or consisting of the amino acid sequence of SEQ ID NO 1, anda heavy chain comprising or consisting of an amino acid sequenceselected from any of SEQ ID NO 3 to 12. Alternatively the antibody has alight chain comprising or consisting of an amino acid sequence selectedfrom SEQ ID NO 13 to 21, and a heavy chain comprising or consisting ofthe amino acid sequence of SEQ ID NO 2. The antibody preferably has twolight chains and two heavy chains.

Also provided is an antibody having a variable region providing bindingspecificity to a target, and a constant region, wherein the constantregion comprises a light chain constant region having the amino acidsequence of residues 109 to 214 of SEQ ID NO 1, and a heavy chainconstant region having the amino acid sequence of residues 121 to 450 ofa sequence selected from SEQ ID NO 3 to 12. Alternatively, the constantregion comprises a light chain constant region having the amino acidsequence of residues 109 to 214 of SEQ ID NO 13 to 21, and a heavy chainconstant region having the amino acid sequence of residues 121 to 450 ofSEQ ID NO 2. Preferably, the antibody comprises a light chain constantregion having the amino acid sequence of residues 109 to 214 of SEQ IDNO 14, and a heavy chain constant region having the amino acid sequenceof residues 121 to 450 of SEQ ID NO 2; or the antibody comprises a lightchain constant region having the amino acid sequence of residues 109 to214 of SEQ ID NO 14, and a heavy chain constant region having the aminoacid sequence of residues 121 to 450 of SEQ ID NO 4; or the antibodycomprises a light chain constant region having the amino acid sequenceof residues 109 to 214 of SEQ ID NO 21, and a heavy chain constantregion having the amino acid sequence of residues 121 to 450 of SEQ IDNO 2.

Also provided is an antibody-drug conjugate (ADC), comprising anantibody according to the first aspect of the invention, conjugated to apayload, preferably a drug payload, such that the DAR is from 1.7-2.2 incase of one mutation on either heavy or light chain and a DAR of 3.4-4.4in case of 2 mutations in light or heavy chain or one in light and onein heavy chain.

The drug payload may be a microtubule disrupting agent, or a DNAmodifying agent. Examples of suitable drug payloads include dolastatin,vedotin, monomethyl auristatin F, maytansinoids including DM1 and DM4,duocarmycin analogs, calicheamicin, pyrrolobenzodiazepines, duocarmycin,centanamycin, irinotecan, and doxorubicin. Other drug payloads may beused.

A yet further aspect of the invention provides an antibody or an ADC asherein described, for use as a therapeutic. The invention also providesa pharmaceutical composition comprising an antibody or an ADC as hereindescribed. A further aspect of the invention provides a method forgenerating an ADC, the method comprising conjugating an antibody asherein described to a drug payload.

Another aspect of the invention provides a method for improving aselected characteristic of a parent antibody after conjugation of theantibody to a payload, wherein the improvement is selected from areduction in loss of monomers, reduction in antibody fragmentation,and/or reduction in antibody aggregation after conjugation, wherein themethod comprises preparing a modified antibody having the amino acidsequence of the parent antibody with one or more substitutions inresidues selected from HC T200, HC V170, HC V176, HC T190, HC S139, HCS160, HC S168, HC S443 HC S445, HC S447, LC S171C, LC T180, LC T206, LCV191, LC S202, LC S203 or or LC S208. The substitution is preferablycysteine, but other mutant residues may be used, in particular lysine,glutamine, or a non-natural amino acid.

Definitions

The following terms used herein are given the following definitions:

By “fragment” is meant a portion of the full size antibody which retainsthe specific binding properties of the antibody. By “derivative” ismeant a modified antibody or antibody fragment having one or morechanges to the peptide sequence, and/or bearing one or more functionalgroups or one or more moieties bound thereto, which retains the specificbinding properties of the antibody. A “derivative” may includepost-translationally modified antibodies.

By “positional numbering”, “sequential numbering” and similar terms ismeant the numbering of the amino acid sequence of the peptide in whichthe first residue at the N terminus is designated residue number 1, andsubsequent residues are sequentially numbered residue 2, 3, 4, etc. Thisis contrasted with Kabat or EU numbering systems for antibodies.

By “site specific conjugation sites” are meant amino acid residueswithin an antibody which are specifically modified in order to permitconjugation of a payload.

By “wild type” is meant an unmodified, naturally occurring, peptide ornucleic acid sequence.

By “parent antibody” is meant an antibody which is used as the basis forpreparing modified antibodies.

By “non-natural amino acid” is meant to an amino acid that is not aproteinogenic amino acid, or a post-translationally modified variantthereof. In particular, the term refers to an amino acid that is not oneof the 20 common amino acids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows levels of expression of candidate antibodies in 25 mlCHOK1SV cultures.

FIG. 2 shows results of SEC-HPLC analysis for % monomer of conjugatedand unconjugated variant antibodies, and % aggregation and fragmentationfor conjugated variants.

FIG. 3 shows extent of biotin-maleimide conjugation to light chains ofantibody variants. Open bars represent unconjugated product, stripedbars represent products with a single conjugated payload, and solid barsrepresent products with two conjugated payloads.

FIG. 4 shows extent of biotin-maleimide conjugation to heavy chains ofantibody variants. Open bars represent unconjugated product, stripedbars represent products with a single conjugated payload, and solid barsrepresent products with two conjugated payloads.

FIG. 5 shows calculated drug antibody ratio for the antibody variants ofFIGS. 3 and 4.

FIG. 6 shows percentage biotin decrease over time for the subset ofantibody variants.

FIG. 7 shows calculated drug to antibody ratio of some of the selectedvariants conjugated to MMAE.

FIG. 8 shows the decrease in cell viability for different cell linesthat have been exposed to different concentrations of the selected ADCover a period of 72 h. PBS=phosphate buffered saline;vc-MMAE=valine-citrulline-MMAE.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed a process for rational design ofmodified antibodies to allow selection of antibodies having desiredproperties for production of antibodies conjugated to payloads such asfor example ADCs (ADC variants). The design process incorporates insilico and in vitro screening steps. Thus, the antibodies of the presentinvention share a number of properties, as will be seen.

To incorporate residues for site specific conjugation, it was decided toreplace native residues with another residue such as cysteine residuesin selected positions of antibody structures. Candidate variants wereanalysed (in silico and in vitro) for desirable properties includingtitre and aggregation, and optionally immunogenicity (in silico only).As an initial proof of concept, Herceptin (trastuzumab) was chosen as amodel antibody, and conjugation optimization and analysis carried outwith biotin maleimide.

Criteria for selecting mutation sites included:

-   -   Residues to be mutated to cysteine (cys) must have similar        physicochemical properties or be a small non-hydrophobic        non-charged residue (ser, val, thr, ala)    -   Residues amenable to be mutated to cysteine must be in constant        regions of either light chain (C_(K), Cλ,) or heavy chain (CH1,        CH2 or CH3) of an antibody or a fragment thereof.    -   In cases where a modified antibody or scaffold is used,        introduced cys should be at a distance>5 Å from any        target-binding interface or domain to minimise risk of        interfering with biological activity of the molecule.    -   Mutations to cys should not create intra-chain hydrogen bonds        leading to the alteration of the local environment and the        properties of the protein    -   Mutations to cys must not be placed in the interfaces between        chains or domains of the antibody (or scaffold). As a general        rule modifications should be at a distance>5 Å from residues        involved in either chain-chain or domain-domain interfaces.    -   Mutations to cys should be at a distance>5 Å from any antibody        native cys and should not interfere with the Fc glycosylation        site (i.e. should be placed at a distance>5 Å from residue        Asn295 where glycosylation occurs)    -   Mutations to cys should not increase the chemical degradation        risk/should not introduce undesired post translational        modifications

Screening of the trastuzumab sequence was then carried out to identifysuitable sites for mutation to another residue such as e.g. cysteine.

The unmodified light chain sequence is:

(SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

The unmodified heavy chain sequence is:

(SEQ ID NO: 2) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEIKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

Ser, Thr, Val, and Ala residues in CH1, CH3, and CL were explored. Thisgave a number of candidates:

Light: SER171, VAL191, SER208, SER182, THR180, THR206, ALA184, SER203,SER202

Heavy: VAL170, VAL176, THR190, SER445, SER443, SER139, SER160, SER447,THR200, SER168.

These mutations were then analysed for desirable properties. Solventaccessibility surface modelling was carried out in silico. DiscoveryStudio (Accelrys Software Inc., Discovery Studio Modeling Environment,Release 4.0, San Diego: Accelrys Software Inc., 2013.) was used tocalculate the Side Chain Solvent Accessibility Surface of the chosenresidues. Solvent accessibility should be greater than 15% (>15%), orgreater than 17% (>17%) to facilitate ‘conjugability’ of the molecule.The percentage side chain solvent accessibility surface is calculated as100 times the side chain solvent accessibility divided by the side chainsolvent accessibility of the fully exposed amino acid residue calculatedusing the extended Ala-X-Ala tripeptide, where X is the residue ofinterest. Side chains with solvent accessibility ratios of equal to orless than 15% (<=15%) or equal to or less than 17% (<=17%) areconsidered buried and not taken into account. The results of the SASmodelling are shown below:

Variant LC % SAS Variant HC % SAS SER171 5.45 VAL170 17.74 VAL191 48.069VAL176 25.179 SER208 48.585 THR190 27.548 SER182 67.216 SER445 33.961THR180 67.518 SER443 46.173 THR206 75.247 SER139 53.401 ALA184 116.94SER160 61.951 SER203 122.689 SER447 72.323 SER202 136.452 THR200 91.354SER168 133.127

Aggregation propensity modelling was also carried out in silico.Aggregation propensity should not be significantly increased by theintroduction of the intended engineered Cys of any of these (potential)ADC variants. This propensity will be calculated based on a Z scorecomparison of the reference molecule and any of the (potential) ADCvariants described above to the distribution of values for a referenceset of the smallest functional domain of the antibody or protein wherethe mutation to Cys is introduced. A mean and standard deviation isdetermined for the reference set. The Z-score is then calculated bysubtracting the reference mean from the target proteins score anddividing by the standard deviation. The result is a zero (0) centredscore where positive values indicated that the target is moreaggregation prone (in this case) than the mean. Targets with a Z-scorewithin (−1, 1) are within the standard deviation of the score within thereference set. The AggreSolve™ in silico platform (Lonza, Basel,Switzerland) comprises a collection of algorithms which, based onsequence and structural parameters, can calculate predictors thatreflect the aggregation propensity of a given polypeptide. Suchpredictors reflect global and local (residue-specific) aggregationpropensities as well as local flexibility and stability.

Difference Antibody Name Z-score (Variant − WT) Trastuzumab Heavy Chain(H) 0.36 H:S160C 0.02 −0.34 H:T190C 0.36 0.00 H:S443C 0.19 −0.17 H:S447C0.19 −0.17 Trastuzumab Light Chain (L) 3.09 L:T180C 2.85 −0.24 L:T206C3.10 0.01 Trastuzumab Heavy Chain constant 0.77 domain 1 (CH1) CH1:S160C0.17 −0.60 CH1:T190C 0.80 0.04 Trastuzumab Heavy Chain constant −0.07domain 3 (CH3) CH3:S443C −0.33 −0.26 CH3:S447C −0.36 −0.29 TrastuzumabLight Chain constant 1.95 domain (CL) CL:T180C 1.66 −0.29 CL:T206C 1.980.03

The AggreSolve Z-score has been calculated for the full lengthTrastuzumab heavy and light chain, as well as for the CH1, CH3, and CLdomains in which the ADC substitutions are located (the minimalfunctional domains).

The boundaries for the CH1, CH3 and CL domains are as per the IMGTdefinition in M. P. Lefranc, C. Pommie, Q. Kaas, E. Duprat, N. Bosc, D.Guiraudou, C. Jean, M. Ruiz, I. Da Piedade, M. Rouard, E. Foulquier, V.Thouvenin, and G. Lefranc. IMGT unique numbering for immunoglobulin andT cell receptor constant domains and Ig superfamily C-like domains.Developmental and comparative immunology 29 (3), 2005.

Following these in silico selection steps, in vitro tests were carriedout on the variants to determine protein yield (in vitro),aggregation/fragmentation, and binding kinetics.

-   -   Protein yield, as estimated by product titre in supernatant and        after protein A purification must be at least 70% or higher of        the parental molecule    -   The percentage of monomer lost after conjugation measured        through Size Exclusion Chromatography HPLC) is preferably ≤35%,        more preferably ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%.    -   Percentage aggregation of the conjugated molecule is preferably        <5%, <10%, <15%, or <20%.    -   Percentage fragmentation of the conjugated antibody antibody is        preferably <5%, <10%, <15%, <20%, <25%, <30%, <32%, <35% or        <40%.    -   The Constant of Dissociation (KD) of the conjugated variants        must be equal to or less than 2 (≤2) orders of magnitude than        the reference standard KD of an unmutated or parent antibody.        For the antibodies described herein the reference standard is        herceptin (trastuzumab), although it will be appreciated that        where a different parent antibody is used, then that parent may        be used as a reference standard.

Systems for cloning and expression of antibodies in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host for small immunoglobulin molecules is E. coli. Theexpression of immunoglobulins, such as antibodies and antibodyfragments, in prokaryotic cells such as E. coli is well established inthe art. Expression in eukaryotic cells in culture is also available tothose skilled in the art as an option for production of aimmunoglobulin. Immunoglobulins, such as antibodies and antibodyfragments, may also be expressed in cell-free systems.

Suitable vectors for the expression of immunoglobulins can be chosen orconstructed, containing appropriate regulatory sequences, includingpromoter sequences, terminator sequences, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate.Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate.For further details see, for example, Molecular Cloning: a LaboratoryManual: 2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in CurrentProtocols in Molecular Biology, Second Edition, Ausubel et al. eds.,John Wiley & Sons, 1992. Nucleic acid encoding a variant immunoglobulinor a CH1, VH and/or VL domain thereof may be contained in a host cell.

Variant antibodies were generated as described for example inWO2011021009A1. In detail: DNA encoding the antibody variants asdescribed herein were chemically synthesized and cloned into a suitablemammalian expression vector. For transient expression experiments heavyand light chain were cloned into separate expression vectors. Forgeneration of cell lines stably expressing a variant antibody heavy andlight chains were cloned into one single expression vector. Eachexpression vector comprises a DNA encoding a signal sequence upstream ofthe heavy chain and the light chain coding regions to enable secretionof the heavy and light chain from the mammalian cells.

For transient expression, CHOK1SV cells were transfected using forexample Lipofectamine with the expression vectors encoding the variantsas described herein. For example in case of variants comprising at leastone mutation in the light chain, an expression vector comprising saidmutation(s) was co-transfected with a vector encoding the unmodifiedheavy chain; in case of variants comprising at least one mutation in theheavy chain, an expression vector comprising said mutation(s) wasco-transfected with a vector encoding the unmodified light chain. 72 hpost-transfection, supernatants were harvested form the transfectedcells, centrifuged and stored at 4° C. prior to purification.

For Large scale production CHOK1SV cells are transfected as describedabove with a single vector comprising modified or unmodified light andheavy chain. Either pools of stably transfected cell are used forfurther experiments or a clonal selection is performed. Supernatants ofsuch stable transfected cells expressing a variant of the presentinvention was harvested and stored at 4° C. prior to purification.

Cell culture supernatants were Protein A purified using HiTrap columns(GE) and stored at 4° C. prior to concentration and buffer exchange.Samples were concentrated by centrifugation at 2000 g 15-20 min.Material was buffer exchanged 4-5 times using formulation buffer (50 mMPhosphate, 100 mM NaCl, pH 7.4). Once buffer exchanged, samples werediluted in formulation buffer to an appropriate working concentration.

Protein Yield Assessment (In Vitro)

The antibody or antibody variant yield is estimated by product titre insupernatant and after protein A purification (e.g. through sandwichELISA, with absorbance at 280 nm, or via HPLC protein A quantification).

FIG. 1 shows levels of expression of each candidate antibody in 25 mlCHOK1SV cultures. All variants show similar levels of expression.

Conjugation

Conjugation was carried out with biotin-maleimide conjugation to freethiol groups by standard techniques Junutula J R et al, NatureBiotechnology 2008, 8, 925-932; Jeffrey S C et al, Bioconjugate Chem.2013, 24, 1256-1263.

For conjugation to a toxin engineered antibodies are e.g reduced with atris(2-carboxyethyl)phosphine (12.5 eq.) for 2 h at 35° C. and pH 7.7.The mixture is buffer exchanged into 50 mM Tris, 5 mM EDTA, pH 7.7.Dehydroascorbic acid (15 eq.) is added and the oxidation reactionallowed to proceed for 3 h at 24° C. N,N-dimethylacetamide is added toreach a concentration of typically between 1 and 5%.

Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatinE (5 eq.) is added and the conjugation reaction allowed to proceed for 1h at 22° C. The reaction is quenched by addition of N-acetyl-cysteine (5eq.). Following 0.5 h incubation at 22° C., the mixture is bufferedexchanged into 1× PBS.

Aggregation Propensity/Fragmentation Assessment (In Vitro)

Before and after conjugation the percentage of monomer lost due toantibody aggregation and/or fragmentation was measured quantitativelyusing Size Exclusion Chromatography HPLC (SEC-HPLC) and qualitativelyusing SDS PAGE. For the latter, each variant antibody was treated withbeta mercaptoethanol, or given no treatment, and size fractionated on aSDS PAGE. There was no apparent aggregation or fragmentation of thevariants visible.

Results from SEC-HPLC analysis of conjugated and unconjugated samplesare shown in FIG. 2, using samples at 1 mg/ml on a Zorbax-250GF column.Surprisingly some of the antibodies of the invention such as for exampleHC S139C, HC V170C, HC S160C, HC T200C, LC V191C, LC T206C, and LC T180Cshowed less monomer lost, and/or decreased amount of antibodyaggregation or fragmentation compared with the parent/unmutatedantibody.

Binding Kinetics Assessment (In Vitro)

Binding kinetics of the variants were also analysed using a quartzcrystal microbalance. ERB2/HER2 Fc chimaera were immobilized to carboxylchip, and three different concentrations of each variant (conjugated andnot conjugated) were tested. The table below summarises the Kd for eachvariant:

K_(D) (nM) Not Conjugated Conjugated Reference Herceptin 3.0 1.63Variant LCHerS208C — — HCHerS443C 4.36 31.13 LCHerS202C 12.22 0.72HCHerT200C 3.52 12.91 HCHerV170C 3.43 2.90 HCHerS447C 3.17 1.34LCHerV191C 4.94 3.56 HCHerS445C 1.27 1.74 HCHerS168C 18.11 1.55HCHerT190C 1.26 2.28 HCHerS139C 3.56 2.44 LCHerT206C 9.41 19.91LCHerT180C 5.19 1.46 HCHerS160C 9.76 1.50 LCHerS182C 5.28 0.008LCHerA184C 1.25 1.04 LCHerS203C — —

Drug to Antibody Ratio (DAR) Assessment (In Vitro)

Finally, the DAR was determined for each of the variants, by liquidchromatography-electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS or LC-ESI-MS). The DAR values for the different variantsmust be >1.7 and <2.2, as there are two site specific conjugation sitesper antibody.

For determining the DAR samples at 1 mg/ml were treated with PNGaseF.Reduced and not-reduced samples were analysed, by RP chromatography,electrospray, and mass spectrometry.

The extent of biotin-maleimide conjugation to light and heavy chains ofthe variants is shown in FIGS. 3 and 4 respectively, with the calculatedDAR for each variant being shown in FIG. 5.

Conjugation and De-Conjugation Assessment (In Vitro)

When stability of the ADC is analysed in vitro, the percentage averageloss of conjugated molecule over a period of 8 days should be <39%.

Six preferred variants were selected, and each of the preferred sixvariants was then further analysed. Conjugate stability (levels ofdeconjugation) was determined for four different concentrations (150ng/ml; 300 ng/ml; 1000 ng/ml and 2000 ng/ml) of each of the finalvariants in human serum at 37 deg C. for 8 days. Samples were taken ondays 0, 2, 4, and 8, and analysed by ELISA.

The percentage biotin decrease over 8 days is shown in FIG. 6.

Each of the final six variants was then ranked for desirablecharacteristics (purity, DAR, deconjugation, and positive environment),and given a score from 6 (=best) to 1 (=worst). The scores were thentotalled, to give an overall score from 4 to 24. This gives anindication of the desirability of each antibody for further development.The scores are shown in the table below.

% Environment Purity DAR Deconjugation Positive Total HCHerS443C 60.83 12.00 6 29 6 LYS 6 19 HCHerS447C 66.94 2 2.09 4 40 1 2 9 HCHerT190C 77.193 1.77 2 37 3 2 10 LCHerT206C 82.7 5 2.13 3 40 1 LYS 6 15 LCHerT180C84.58 6 1.76 1 32 5 2 14 HCHerS160C 81.03 4 1.96 5 32 5 2 16

Although the antibody variants can be ranked in this way, as each of thefinal six has been through the initial selection process, they can allbe said to have desirable characteristics for development as an ADC. Inparticular, not every antibody will make it through subsequent drugdevelopment processes and in vivo testing, so it is beneficial to beable to generate a selection of candidates. Furthermore, other variantsnot selected for the final six, such as the remaining variants disclosedherein, may also have beneficial properties and so may be considereduseful for further investigation.

The six final variants were: four heavy chain (S160C, T190C, S443C,S447C), and two light chain (T180C, or T206C) variants. As a result ofthe sequential method of selection all final variants can be expected toshare a number of specific properties (or design criteria): Stability;low aggregation; low chemical degradation risk; low undesired posttranslational modifications; structural stability preserved;productivity; suitability for being conjugated; and biological activity.

The values for each tested variant are shown in the table below; the sixfinal selected variants are highlighted.

Prot A HPLC SEC HPLC K_(D) (nM) LC MS Light Chain mg/L % Monomer conj.Ab NOT CONJUGATED CONJUGATED Unmodified +1 Conjugate +2 ConjugatesHerceptin 55.13 78.53 3.02 1.63 100% 0% 0% LCHerS208C 48.11 54.06 — — —— — HCHerS443C 54.96 60.83 4.36 31.13 89% 11% 0% LCHerS202C 55.00 69.0012.22 0.72 32% 60% 7% HCHerT200C 58.83 94.94 3.52 12.91 100% 0% 0%HCHerV170C 55.42 93.54 3.43 2.90 100% 0% 0% HCHerS447C 49.11 66.94 3.171.34 81% 19% 0% LCHerV191C 56.37 95.73 4.94 3.56 100% 0% 0% HCHerS445C57.93 77.22 1.27 1.74 78% 22% 0% HCHerS168C 57.68 78.01 18.11 1.55 93%7% 0% HCHerT190C 46.22 77.19 1.26 2.28 94% 6% 0% HCHerS139C 60.58 80.273.56 2.44 84% 16% 0% LCHerT206C 53.83 82.70 9.41 19.91 4% 88% 8%LCHerT180C 46.87 84.58 5.19 1.46 16% 81% 4% HCHerS160C 55.47 81.03 9.761.50 95% 5% 0% LCHerS182C 51.18 73.15 5.28 8 × 10−3 38% 62% 0%LCHerA184C 60.11 80.20 1.25 1.04 86% 14% 0% LCHerS203C 53.61 58.22 — — —— — LC MS Heavy Chain Unmodified +1 Conjugate +2 Conjugates +3Conjugates DAR Herceptin 100% 0% 0% 0% 0.00% LCHerS208C — — — — —HCHerS443C 11% 89% 0% 0% 2.00% LCHerS202C 95% 5% 0% 0% 1.60% HCHerT200C100% 0% 0% 0% 0.00% HCHerV170C 100% 0% 0% 0% 0.00% HCHerS447C 15% 85% 0%0% 2.09% LCHerV191C 100% 0% 0% 0% 0.00% HCHerS445C 22% 35% 34%  8% 3.01%HCHerS168C 39% 61% 0% 0% 1.36% HCHerT190C 18% 82% 0% 0% 1.77% HCHerS139C22% 52% 22%  4% 2.47% LCHerT206C 97% 3% 0% 0% 2.13% LCHerT180C 100% 0%0% 0% 1.76% HCHerS160C 7% 93% 0% 0% 1.96% LCHerS182C 100% 0% 0% 0% 1.25%LCHerA184C 100% 0% 0% 0% 0.28% LCHerS203C — — — — —

Subsequently the final six variants were conjugated to MonomethylAuristatin E (MMAE) by standard techniques. The conjugation methodfollows broadly methods described above. The DAR for the selectedvariants was determined as described above. An example of the results isshown in FIG. 7. Cond 1-3 represent minor variants in the conjugationprocedure with parameters varied to try to optimise the DAR; thereduction time and temperature for antibodies prior to conjugation werevaried in each of conditions 1-3:

Condition 1: reduction at 35° C. for 2 h (as described above)

Condition 2: reduction at 25° C. for 2 h

Condition 3: reduction at 35° C. for 1 h.

A double mutant (DM) combining LC T180C and HC S160C was also tested todetermine aggregation propensity and DAR data, using the same techniquesas described above. The results are shown in the following tables:

DM Aggregation Data from the Transient Transfections

using size-exclusion chromatography SEC

Relative % by SEC Species Before conjugation After conjugation Purity,main peak 95.4 96.1 High molecular weight forms 3.0 3.0 Low molecularweight forms 1.6 0.9

DM DAR Data from the Transient Transfections

using PLRP HPLC

or ESI-MS methods

Variant PLRP Intact mass DM 3.77 3.97

In Vitro Toxicity Tests

After MMAE conjugation the ADC variants were tested for in vitrocytotoxicity. The analysis was carried out by standard techniques(Andreotti, P. E. et al. Cancer Res 1995, 55, 5276-82; Gerhardt, R. T.et al. Am. J. Obstet. Gynecol 1991 165, 245-55). The cells chosen forthe assay were based on Neve R. M. et al. Cancer Cell 2006 10, 515-527.

Assay schematics:

Day 1: Seed three 96-well plates each of SKBR3 cells (5 k/well) in media(McCoy5A+10% FBS+1×Pen/Strep), BT474 cells (8 k/well) in media(DMEM/F12+10% FBS+1×Pen/Strep), and MCF7 cells (4 k/well) in media(RPMI+10% FBS+1×Pen/Strep). Incubate in 37° C. humidified CO2 incubatorfor 18 hrs.

Day 2: Prepare ADC variants sample dilutions. Make the initial 667 nMworking stocks of these samples in RPMI media with 10% FBS. Then prepare⅓ serial dilution from 667 nM to 11 pM in media. Add 5 ul of thedilution into each well of ˜100 ul cells. Final sample concentrationsrange from 33.3 nM to 0.56 pM (as ⅓ serial dilutions). Incubate at 37°C. in a humidified CO2 incubator for 72 hrs.

Day 4: Evaluate the plates under a microscope

Day 5: Determine cell viability using Cell-Titer Glo reagent:

-   -   Aspirate the media from the 96-well plate.    -   Add 100 ul of Cell-Titer Glo reagent (Promega Inc.) in each        well. Incubate at room temperature for 10 min.    -   Determine luminescence using Tecan Ultra plate reader.    -   Analyze andplot data either as Percent Viability vs.        Concentration (nM), or as random luminesce.

An example of the results is shown in FIG. 8. As can be seen in FIG. 8,ADC variants HC S443C and LC T180C reduce the viability of SKBR3 cellsand BT474 cells by 50% at very low concentrations, whereas these ADCvariants do not show an effect over 72 hours in less responsive cellslike MCF7.

The full sequences of the variant chain of each of the variantsdescribed herein are shown below. These show only the variant chain; theother chain will be the same as the unmodified trastuzumab sequence(that is, SEQ ID No 1 (LC) or 2 (HC)).

Heavy chains: >HCherS139C (SEQ ID No 3)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTCGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherS160C (SEQ ID No 4)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVCWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherS168C (SEQ ID No 5)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherV170C (SEQ ID No 6)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGCHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherV176C (SEQ ID No 7)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPACLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherT190C (SEQ ID No 8)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVCVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherT200C (SEQ ID No 9)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >HCherS443C (SEQ ID No 10)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKCLSLSPGK >HCherS445C (SEQ ID No 11)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK >HCherS447C (SEQ ID No 12)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLCPGKLight chains: >LCherS171C (SEQ ID No 13)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDCTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >LCherT180C (SEQ ID No 14)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLCLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >LCherS182C (SEQ ID No 15)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLCKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >LCherA184C (SEQ ID No 16)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKCDYEKHKVYACEVTHQGLSSPVTKSFNRGEC >LCherV191C (SEQ ID No 17)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKCYACEVTHQGLSSPVTKSFNRGEC >LCherS202C (SEQ ID No 18)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREADVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLCSPVTKSFNRGEC >LCherS203C (SEQ ID No 19)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSCPVTKSFNRGEC >LCherT206C (SEQ ID No 20)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCKSFNRGEC >LCherS208C (SEQ ID No 21)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKCFNRGEC

It will be appreciated that a similar selection and screening processmay be used to develop other variant antibodies, not only those based ontrastuzumab, and further that it may be expected that variants of theseother antibodies having the same constant region mutations as identifiedherein would also be expected to have similar desirable properties.

The invention claimed is:
 1. An antibody, or a fragment or derivativethereof, having a variable region which binds a target molecule, and aconstant region, wherein the constant region comprises one or moremutations introducing a site specific conjugation site selected so as topermit conjugation of the antibody, fragment, or derivative to apayload; wherein the antibody comprises the amino acid sequence of SEQID NO: 40, where X is C; and wherein the antibody further comprises oneor more amino acid sequences selected from the group consisting of SEQID NO: 22-39, where each X is C.
 2. The antibody of claim 1, wherein theantibody is selected from the group comprising IgG1, IgG2, IgG3, andIgG4.
 3. The antibody of claim 1, wherein the antibody is selected fromthe group consisting of Fabs, bi specific antibody fragments (tandemscFv-Fc, scFv-Fc knobs-into-holes, scFv-Fc-scFv, F(ab′)2, Fab-scFv,(Fab′ scFv)2, scDiabody-Fc, or scDiabody-CH3), IgG-based bispecificantibodies (Hybrid hybridoma, Knobs-into-holes with common light chain,Two-in-one IgG, Dual V domain IgG, IgG-scFv, scFv-IgG, IgG-V, V-IgG),minibody, tribi-minibody, nanobodies, and di-diabody.
 4. The antibody ofclaim 1, wherein the antibody is human, humanised, or chimeric.
 5. Theantibody of claim 1, which lacks one or more Fc effector functions. 6.The antibody of claim 1, further comprising a linker, wherein the linkeris selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP),valine-citrulline (val-cit), alanine-phenylalanine (ala-phe),p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1carboxylate (SMCC), N-Succinimidyl, (4-iodo-acetyl) aminobenzoate(SIAB), SPDB, hydrazone, maleimidocaproyl and6-maleimidocaproyl-valine-citrulline-p-aminobenyloxycarbonyl(MC-vc-PAB); or is a branched linker which comprises a peptide chain andis derived from o-hydroxy p-amino benzylic alcohol, wherein the peptidechain is connected to the phenyl ring via the p-amino group, the payloadis connected to the phenyl ring via the benzylic alcohol moiety, and theantibody is connected to the phenyl ring via the o-hydroxy group.
 7. Animmunoconjugate comprising an antibody according to claim 1, a payload,and a linker joining the payload to the antibody.
 8. The immunoconjugateof claim 7, wherein the linker is selected from 6-maleimidocaproyl (MC),maleimidopropanoyl (MP), valine-citrulline (val-cit),alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB),N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), N-Succinimidyl,(4-iodo-acetyl) aminobenzoate (SIAB), SPDB, hydrazone, maleimidocaproyland 6-maleimidocaproyl-valine-citrulline-p-aminobenyloxycarbonyl(MC-vc-PAB); or is a branched linker which comprises a peptide chain andis derived from o-hydroxy p-amino benzylic alcohol, wherein the peptidechain is connected to the phenyl ring via the p-amino group, the payloadis connected to the phenyl ring via the benzylic alcohol moiety, and theantibody is connected to the phenyl ring via the o-hydroxy group, andwherein the payload is selected from the group consisting of aradionuclide, a chemotherapeutic agent, a cytotoxic agent, a microbialtoxin, a plant toxin, a polymer, a carbohydrate, a cytokine, afluorescent label, a luminescent label, an enzyme-substrate label, anenzyme, a peptide, a peptidomimetic, a nucleotide, an siRNA, a microRNA,an RNA mimetic, and an aptamer.
 9. The immunoconjugate of claim 7,wherein the payload is selected from the group consisting of 90Y, 131I,67Cu, 177Lu, 213Bi, 211At, dolastatin, vedotin, monomethyl auristatinF(MMAF), monomethyl auristatin E (MMAE); maytansinoids including DM1 andDM4, duocarmycin, duocarmycin analogs, calicheamicin,pyrrolobenzodiazepines (PBD), centanamycin, irinotecan, and doxorubicin,alpha-amanitin, melatonin, membrane disrupting peptide, Pseudomonasexotoxin A, Diphtheria toxin, ricin, polyethylene glycol, hydroxyethylstarch, and a mannosyl residue.
 10. A pharmaceutical compositioncomprising an antibody according to claim 1, and a pharmaceuticallyacceptable diluent, carrier or excipient.
 11. A method for generating animmunoconjugate, the method comprising conjugating an antibody accordingto claim 1 to a payload.
 12. The antibody of claim 1 prepared by aprocess comprising: mutagenizing a nucleic acid sequence of a parentantibody by replacing one or more amino acid residues with a mutantresidue to encode the antibody; expressing the mutant antibody; andisolating the mutant antibody.
 13. The antibody of claim 12, the processfurther comprising: reacting the mutant antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, antibody; andmeasuring the binding of the affinity labelled antibody to a capturemedia.
 14. An isolated or recombinant polynucleotide encoding theantibody of claim
 1. 15. A vector comprising the polynucleotide of claim14, wherein the vector further comprises an inducible promoter operablylinked to the polynucleotide.
 16. A host cell comprising the vector ofclaim
 15. 17. A method of producing an antibody comprising: providing aculture medium comprising the host cell of claim 16; and placing theculture medium in conditions under which the antibody is expressed, andoptionally (c) isolating the antibody.
 18. The antibody of claim 2,wherein the constant region comprises at least a portion of an IgG1constant region.
 19. The antibody of claim 18, wherein the constantregion comprises one or more of the Ck, CH1 and CH3 domains of the IgG1constant region.
 20. The antibody of claim 4, wherein the variableregions of the antibody rare selected from the variable regions of oneof Abciximab; Rituximab; Basiliximab; Daclizumab; Palivizumab;Infliximab; Trastuzumab; Alemtuzumab; Adalimumab; Efalizumab; Cetuximab;Ibritumomab; Omalizumab; Bevacizumab; Ranibizumab; Golimumab;Canakinumab; Ustekinumab; Tocilizumab; Ofatumumab; Belimumab;Ipilimumab; Brentuximab; Pertuzumab; Raxibacumab; Vedolizumab;Ramucirumab; Obinutuzumab; Siltuximab; Secukinumab; or Dinutuximab. 21.The antibody of claim 5, wherein the antibody lacks ADCC activity or hasincreased ADCC activity.
 22. The immunoconjugate of claim 7, wherein thepayload is a microtubule disrupting agent, or a DNA modifying agent. 23.The antibody of claim 12, wherein the antibody is expressed in amammalian or an insect cell.